Systematics of the marine microfilamentous green algae Uronema curvatum and Urospora microscopica (Chlorophyta)

The microfilamentous green alga Uronema curvatum is widely distributed along the western and eastern coasts of the north Atlantic Ocean where it typically grows on crustose red algae and on haptera of kelps in subtidal habitats. The placement of this marine species in a genus of freshwater Chlorophyceae had been questioned. Molecular phylogenetic analysis of nuclear-encoded small and large subunit rDNA sequences reveal that U. curvatum is closely related to the ulvophycean order Cladophorales, with which it shares a number of morphological features, including a siphonocladous level of organization and zoidangial development. The divergent phylogenetic position of U. curvatum, sister to the rest of the Cladophorales, along with a combination of distinctive morphological features, such as the absence of pyrenoids, the diminutive size of the unbranched filaments and the discoid holdfast, warrants the recognition of a separate genus, Okellya, within a new family of Cladophorales, Okellyaceae. The epiphytic Urospora microscopica from Norway, which has been allied with U. curvatum, is revealed as a member of the cladophoralean genus Chaetomorpha and is herein transferred to that genus as C. norvegica nom. nov.     

A phylogeny of Diprotodontia (Marsupialia) based on sequences for five nuclear genes

Even though the marsupial order Diprotodontia is one of the most heavily studied groups of Australasian marsupials, phylogenetic relationships within this group remain contentious. The more than 125 living species of Diprotodontia can be divided into two main groups: Vombatiformes (wombats and koalas) and Phalangerida. Phalangerida is composed of the kangaroos (Macropodidae, Potoroidae, and Hypsiprymnodontidae) and possums (Phalangeridae, Burramyidae, Petauridae, Pseudocheiridae, Tarsipedidae, and Acrobatidae). Much of the debate has focused on relationships among the families of possums and whether possums are monophyletic or paraphyletic. A limitation of previous investigations is that no study to date has investigated diprotodontian relationships using all genera. Here, we examine diprotodontian interrelationships using a nuclear multigene molecular data set representing all recognized extant diprotodontian genera. Maximum parsimony, maximum likelihood, and Bayesian methods were used to analyze sequence data obtained from protein-coding portions of ApoB, BRCA1, IRBP, Rag1, and vWF. We also applied a Bayesian relaxed molecular clock method to estimate times of divergence. Diprotodontia was rooted between Vombatiformes and Phalangerida. Within Phalangerida, the model-based methods strongly support possum paraphyly with Phalangeroidea (Burramyidae + Phalangeridae) grouping with the kangaroos (Macropodiformes) to the exclusion of Petauroidea (Tarsipedidae, Acrobatidae, Pseudocheiridae, and Petauridae). Within Petauroidea, Tarsipedidae grouped with both Petauridae and Pseudocheiridae to the exclusion of Acrobatidae. Our analyses also suggest that the diprotodontian genera Pseudochirops and Strigocuscus are paraphyletic and diphyletic, respectively, as currently recognized. Dating analyses suggest Diprotodontia diverged from other australidelphians in the late Paleocene to early Eocene with all interfamilial divergences occurring prior to the early Miocene except for the split between the Potoroidae and Macropodidae, which occurred sometime in the mid-Miocene. Ancestral state reconstructions using a Bayesian method suggest that the patagium evolved independently in the Acrobatidae, Petauridae, and Pseudocheiridae. Ancestral state reconstructions of ecological venue suggest that the ancestor of Diprotodontia was arboreal. Within Diprotodontia, the common ancestor of Macropodidae was reconstructed as terrestrial, suggesting that tree kangaroos (Dendrolagus) are secondarily arboreal.     

Ultrastructure and phylogenetic relationships of the unicellular green algae Ignatius tetrasporus and Pseudocharacium americanum (Chlorophyta)

The phylogenetic relationships of two unicellular green algae, Ignatius tetrasporus Bold et MacEntee and Pseudocharacium americanum Lee et Bold were investigated by ultrastructural and molecular methods. The zoospores from both species were covered neither by scales nor cell walls. The flagellar apparatus of the zoospores commonly included these features: the upper basal bodies were displaced counterclockwise in half to two‐thirds of the basal body diameter and did not overlap with each other; the lower basal bodies were directly opposed or slightly displaced clockwise; the distal fiber had gently sigmoid central striations; terminal caps were absent from the ends of the basal bodies; a V‐shaped proximal sheath extended from the upper basal bodies; a posterior fiber lay between the opposite lower basal bodies; and the coarsely striated band linked the sinister rootlet to the lower basal body. The suite of these features was not identical to that of any other quadriflagellate swimming cells, but some features including the lower basal body orientation, the striated distal fiber, and the coarsely striated fiber resemble those of the several organisms of the Siphonocladales sensu Floyd and O’Kelly. Phylogenetic analysis using 18S rDNA sequence data revealed that I. tetrasporus and P. americanum formed a monophyletic clade within the clade of Ulvophyceae sensu López‐Bautista and Chapman, but was not nested within any of the orders of the class that were examined.     

Molecular,biochemical and morphological data suggest an affiliation of Spongiochrysis hawaiiensis with the Trentepohliales (Ulvophyceae,Chlorophyta)

The aeroterrestrial, unicellular green alga Spongiochrysis hawaiiensis had been included in the ulvophycean order Cladophorales based on small subunit (SSU) rDNA sequence data, and represents so far the only fully terrestrial member of this order. Other characteristics of S. hawaiiensis that are atypical for Cladophorales include the presence of large amounts of carotenoids and a budding‐like mode of cell division. As the position of this terrestrial, unicellular alga in an order of aquatic, multicellular green algae is unusual, we re‐evaluated the phylogenetic relationships of this enigmatic organism based on supplementary SSU rDNA sequences as well as novel large ribosomal subunit (LSU) rDNA and internal transcribed spacer (ITS rDNA) sequences. Additionally, we examined several morphological characters of S. hawaiiensis, as well as low molecular weight carbohydrate (LMWC) patterns of S. hawaiiensis and members of the Cladophorales and Trentepohliales as potential chemotaxonomic markers. We found S. hawaiiensis to be uninucleate. The analysis of the LMWC content detected the presence of the polyol erythritol in S. hawaiiensis and in the Trentepohliales, while this compound was missing in the Cladophorales. The phylogenetic analyses of the novel sequences placed S. hawaiiensis in the terrestrial Trentepohliales. This placement is supported by the aeroterrestrial habitat, the presence of large amounts of carotenoids, the uninucleate cells, and the presence of the polyol erythritol as a protective compound against water loss.     

Higher plant origins and the phylogeny of green algae

Summary 5S rRNA sequences from six additional green algae lend strong molecular support for the major outlines of higher plant and green algae phylogeny that have been proposed under varying naming conventions by several authors. In particular, the molecular evidence now available unequivocally supports the existence of at least two well-separated divisions of the Chlorobionta: the Chlorophyta and the Streptophyta (i.e., charophytes) (according to the nomenclature of Bremer). The chlamydomonad 5S rRNAs are, however, sufficiently distinct from both clusters that it may ultimately prove preferable to establish a third taxon for them. In support of these conclusions 5S rRNA sequence data now exist for members of four diverse classes of chlorophytes. These sequences all exhibit considerably more phylogenetic affinity to one another than any of them show toward members of the other cluster, the Streptophyta, or the twoChlamydomonas strains. Among the Charophyceae, new 5S rRNA sequences are provided herein for three genera,Spirogyra, Klebsormidium, andColeochate. All of these sequences and the previously publishedNitella sequence show greater resemblance among themselves and to the higher plants than they do to any of the other green algae examined to date. These results demonstrate that an appropriately named taxon that includes these green algae and the higher plants is strongly justified. The 5S rRNA data lack the resolution needed, however, to unequivocally determine which of several subdivisions of the charophytes is the sister group of the land plants. The evolutionary diversity ofChlamydomonas relative to the other green algae was recognized in earlier 5S rRNA studies but was unanticipated by ultrastructural work. These new data provide further evidence for the relative uniqueness of the chlamydomonads and are discussed further.     

Non-calcified macroalgae from the lower Famennian (Upper Devonian) of the Holy Cross Mountains,Poland

Non-calcified algal remains were collected from the lower Famennian deposits at the Kowala quarry in the Holy Cross Mountains, central Poland. Each specimen is represented solely by vegetative organs. On the basis of the material collected, a new algal genus and species has been formally erected. Vittella dixii nov. gen., nov. sp. (Order ?Bryopsidales) includes thalli consisting of long bundles of hair-like filament structures arranged in parallel fashion to the thallus length. Some other algal remains are regarded as Inocaulis-like forms. They possess long, flat blades with entire margins, and are ornamented with very dense, hair-like projections. The rest of the specimens found so far has been illustrated and tentatively classified as probably belonging to either Bryopsidales or Dasycladales algae. The exceptional preservation of the non-calcified algal thalli investigated suggests that special taphonomic conditions have prevailed during sedimentation of the lower part of the Famennian Kowala section. These may have involved rather rapid burial in oxygen-restricted (dysoxic and anoxic) sea-bottom environment, inhibiting the activities of scavengers and bioturbators, as well as oxidative degradation. The preservation of the algae, together with sedimentological and geochemical characteristics of the host rocks indicate that they rather represent parautochthonous assemblage, buried close to their natural habitat.     

Dating the origin of the major lineages of Branchiopoda

Despite the well-established phylogeny and good fossil record of branchiopods, a consistent macro-evolutionary timescale for the group remains elusive. This study focuses on the early branchiopod divergence dates where fossil record is extremely fragmentary or missing. On the basis of a large genomic dataset and carefully evaluated fossil calibration points, we assess the quality of the branchiopod fossil record by calibrating the tree against well-established first occurrences, providing paleontological estimates of divergence times and completeness of their fossil record. The maximum age constraints were set using a quantitative approach of Marshall (2008). We tested the alternative placements of Yicaris and Wujicaris in the referred arthropod tree via the likelihood checkpoints method. Divergence dates were calculated using Bayesian relaxed molecular clock and penalized likelihood methods. Our results show that the stem group of Branchiopoda is rooted in the late Neoproterozoic (563 ± 7 Ma); the crown-Branchiopoda diverged during middle Cambrian to Early Ordovician (478–512 Ma), likely representing the origin of the freshwater biota; the Phyllopoda clade diverged during Ordovician (448–480 Ma) and Diplostraca during Late Ordovician to early Silurian (430–457 Ma). By evaluating the congruence between the observed times of appearance of clade in the fossil record and the results derived from molecular data, we found that the uncorrelated rate model gave more congruent results for shallower divergence events whereas the auto-correlated rate model gives more congruent results for deeper events.     

Phylogenetic position of the coral symbiont Ostreobium (Ulvophyceae) inferred from chloroplast genome data

The green algal genus Ostreobium is an important symbiont of corals, playing roles in reef decalcification and providing photosynthates to the coral during bleaching events. A chloroplast genome of a cultured strain of Ostreobium was available, but low taxon sampling and Ostreobium's early‐branching nature left doubt about its phylogenetic position. Here, we generate and describe chloroplast genomes from four Ostreobium strains as well as Avrainvillea mazei and Neomeris sp., strategically sampled early‐branching lineages in the Bryopsidales and Dasycladales respectively. At 80,584 bp, the chloroplast genome of Ostreobium sp. HV05042 is the most compact yet found in the Ulvophyceae. The Avrainvillea chloroplast genome is ~94 kbp and contains introns in infA and cysT that have nearly complete sequence identity except for an open reading frame (ORF) in infA that is not present in cysT. In line with other bryopsidalean species, it also contains regions with possibly bacteria‐derived ORFs. The Neomeris data did not assemble into a canonical circular chloroplast genome but a large number of contigs containing fragments of chloroplast genes and showing evidence of long introns and intergenic regions, and the Neomeris chloroplast genome size was estimated to exceed 1.87 Mb. Chloroplast phylogenomics and 18S nrDNA data showed strong support for the Ostreobium lineage being sister to the remaining Bryopsidales. There were differences in branch support when outgroups were varied, but the overall support for the placement of Ostreobium was strong. These results permitted us to validate two suborders and introduce a third, the Ostreobineae.     

Mitochondrial genomes of the green macroalga Ulva pertusa (Ulvophyceae,Chlorophyta): novel insights into the evolution of mitogenomes in the Ulvophyceae

To further understand the trends in the evolution of mitochondrial genomes (mitogenomes or mtDNAs) in the Ulvophyceae, the mitogenomes of two separate thalli of Ulva pertusa were sequenced. Two U. pertusa mitogenomes (Up1 and Up2) were 69,333 bp and 64,602 bp in length. These mitogenomes shared two ribosomal RNAs (rRNAs), 28 transfer RNAs (tRNAs), 29 protein‐coding genes, and 12 open reading frames. The 4.7 kb difference in size was attributed to variation in intron content and tandem repeat regions. A total of six introns were present in the smaller U. pertusa mtDNA (Up2), while the larger mtDNA (Up1) had eight. The larger mtDNA had two additional group II introns in two genes (cox1 and cox2) and tandem duplication mutations in noncoding regions. Our results showed the first case of intraspecific variation in chlorophytan mitogenomes and provided further genomic data for the undersampled Ulvophyceae.     

Repeated evolution and reversibility of self‐fertilization in the volvocine green algae*

Outcrossing and self‐fertilization are fundamental strategies of sexual reproduction, each with different evolutionary costs and benefits. Self‐fertilization is thought to be an evolutionary “dead‐end” strategy, beneficial in the short term but costly in the long term, resulting in self‐fertilizing species that occupy only the tips of phylogenetic trees. Here, we use volvocine green algae to investigate the evolution of self‐fertilization. We use ancestral‐state reconstructions to show that self‐fertilization has repeatedly evolved from outcrossing ancestors and that multiple reversals from selfing to outcrossing have occurred. We use three phylogenetic metrics to show that self‐fertilization is not restricted to the tips of the phylogenetic tree, a finding inconsistent with the view of self‐fertilization as a dead‐end strategy. We also find no evidence for higher extinction rates or lower speciation rates in selfing lineages. We find that self‐fertilizing species have significantly larger colonies than outcrossing species, suggesting the benefits of selfing may counteract the costs of increased size. We speculate that our macroevolutionary results on self‐fertilization (i.e., non‐tippy distribution, no decreased diversification rates) may be explained by the haploid‐dominant life cycle that occurs in volvocine algae, which may alter the costs and benefits of selfing.     

Molecular phylogeny of the hexagrammid fishes using a multi-locus approach

Ideally, organisms are grouped into monophyletic assemblages reflecting their evolutionary histories. Single (molecular) markers can reflect the evolutionary history of the marker, rather than the species in question, therefore, phylogenetic relationships should be inferred from adequate sampling of characters. Because the use of multiple loci greatly improves the resolving power of the molecular assay, we constructed a molecular phylogeny of the family Hexagrammidae based on six loci, including two mitochondrial and four nuclear loci. The resulting molecular phylogeny, from the combined data, was significantly different from the morphological topology suggested by Shinohara [Memoirs of the Faculty of Fisheries, Hokkaido University 41 (1994) 1]. Our data support a monophyletic assemblage for the genera Hexagrammos and Pleurogrammus. However, other taxa traditionally included in the family Hexagrammidae did not form a monophyletic assemblage. The monotypic genus Ophiodon was more closely associated with cottids than with other hexagrammids. Our data concur with the morphological topology in that the genera Zaniolepis and Oxylebius formed a monophyletic clade, which was distinct and basal to the remaining hexagrammids, seven cottids and one agonid.     

Chloroplast genome evolution and phylogeny of the early-diverging charophycean green algae with a focus on the Klebsormidiophyceae and Streptofilum

The Klebsormidiophyceae are a class of green microalgae observed globally in both freshwater and terrestrial habitats. Morphology-based classification schemes of this class have been shown to be inadequate due to the simple morphology of these algae, the tendency of morphology to vary in culture versus field conditions, and rampant morphological homoplasy. Molecular studies revealing cryptic diversity have renewed interest in this group. We sequenced the complete chloroplast genomes of a broad series of taxa spanning the known taxonomic breadth of this class. We also sequenced the chloroplast genomes of three strains of Streptofilum, a recently discovered green algal lineage with close affinity to the Klebsormidiophyceae. Our results affirm the previously hypothesized polyphyly of the genus Klebsormidium as well as the polyphyly of the nominal species in this genus, K. flaccidum. Furthermore, plastome sequences strongly support the status of Streptofilum as a distinct, early-diverging lineage of charophytic algae sister to a clade comprising Klebsormidiophyceae plus Phragmoplastophyta. We also uncovered major structural alterations in the chloroplast genomes of species in Klebsormidium that have broad implications regarding the underlying mechanisms of chloroplast genome evolution.     

Identification of teratogenic polymethoxy-1-alkenes from Cylindrospermopsis raciborskii,and taxonomically diverse freshwater cyanobacteria and green algae

Cylindrospermopsis raciborskii is among the most commonly recognized toxigenic cyanobacteria associated with harmful algal blooms (HAB) in freshwater systems, and specifically associated with multiple water-soluble toxins. Lipophilic metabolites from C. raciborskii, however, were previously shown to exert teratogenicity (i.e. inhibition of vertebrate development) in the zebrafish (Danio rerio) embryo model, specifically suggesting the presence of additional bioactive compounds unrelated to the currently known toxins. In the present study, a series of known teratogenic polymethoxy-1-alkenes (PMA) were identified, purified and chemically characterized from an otherwise well-characterized strain of toxigenic C. raciborskii. Although PMA have been previously identified in other cyanobacteria, this is the first time they have been identified from this recognized HAB species. Following their identification from C. raciborskii, the taxonomic distribution of the PMA was additionally investigated by chemical screening of a freshwater algal (i.e. cyanobacteria, green algal) culture collection. Screening suggests that these compounds are distributed among phylogenetically diverse taxa. Furthermore, parallel screening of the algal culture collection, using the zebrafish embryo model of teratogenicity, the presence of PMA was found to closely correlate with developmental toxicity of these diverse algal isolates. Taken together, the data suggest PMA contribute to the toxicity of C. raciborskii, as well as apparently several other taxonomically disparate cyanobacterial and green algal genera, and may, accordingly, contribute to the toxicity of diverse freshwater HAB.     

Aragonitic dasycladalean algae from the Upper Permian of Sichuan, China

Exceptionally well-preserved specimens of Mizzia yabei (Karpinsky) were discovered in bituminous packstone intercalations of the early Upper Permian Maokou Formation of Anxian County (Sichuan, China). Staining techniques and x-ray diffraction reveal that the organic encrustation around the central stem and the lateral branches consists of aragonite. Average aragonite contents range between 27% and 40%. The bulk of the remaining 60–73% calcite represents cement. The total Sr-content is 3, 400 ppm, indicating 8,000–10,000 ppm for the aragonite fraction. The organically precipitated aragonite was deposited as closely packed fibres, 10–20 μm long and 0.5–1 μm wide. The fibres are arranged in clinogonal (water-jet) fascicles, which diverge towards the central (spar-filled) stem. This microstructure shows that the mineralization of the mucilage started at the periphery of the extracellular encrustation and progressed toward its interior. Diagenetic alterations of the original aragonite include various stages of breakdown of the fibres into 0.5–1 μm large granules (micritization), minor syntaxial growth of the fibres and local transformation of acicular aragonite into neomorphic calcite. The dasycladaleans from the Permian of Sichuan appear to be by far the oldest representatives of this order with preserved aragonitic mineralogy and microstructure. Comparably well-preserved examples are only known from Eocene and younger deposits.     

Molecular phylogeny and taxonomic revision of the genus Wittrockiella (Pithophoraceae,Cladophorales), including the descriptions of W. australis sp. nov. and W. zosterae sp. nov.

Wittrockiella is a small genus of filamentous green algae that occurs in habitats with reduced or fluctuating salinities. Many aspects of the basic biology of these algae are still unknown and the phylogenetic relationships within the genus have not been fully explored. We provide a phylogeny based on three ribosomal markers (ITS, LSU, and SSU rDNA) of the genus, including broad intraspecific sampling for W. lyallii and W. salina, recommendations for the use of existing names are made, and highlight aspects of their physiology and life cycle. Molecular data indicate that there are five species of Wittrockiella. Two new species, W. australis and W. zosterae, are described, both are endophytes. Although W. lyallii and W. salina can be identified morphologically, there are no diagnostic morphological characters to distinguish between W. amphibia, W. australis, and W. zosterae. A range of low molecular weight carbohydrates were analyzed but proved to not be taxonomically informative. The distribution range of W. salina is extended to the Northern Hemisphere as this species has been found in brackish lakes in Japan. Furthermore, it is shown that there are no grounds to recognize W. salina var. kraftii, which was described as an endemic variety from a freshwater habitat on Lord Howe Island, Australia. Culture experiments indicate that W. australis has a preference for growth in lower salinities over full seawater. For W. amphibia and W. zosterae, sexual reproduction is documented, and the split of these species is possibly attributable to polyploidization.